“Eco-friendly HPLC method for analysis of dipyrone and hyoscine in different matrices with biomonitoring”

A selective, precise, and accurate reversed HPLC method has been developed and validated for simultaneous separation and determination of two veterinary drugs, dipyrone and hyoscine, in their combined dosage form in the presence of their official impurities, namely 4-aminoantipyrine and tropic acid, in addition to the formulated preservative: phenol. The linearity range was found to be (1.00–35.00 µg/mL) for dipyrone and (2.50–50.00 µg/mL) for hyoscine. It exhibited a satisfactory linearity regression R (0.9999) for both drugs with LOD 0.22 µg/mL and 0.72 µg/mL and LOQ 0.65 µg/mL and 2.19 µg/mL for dipyrone and hyoscine, respectively. Additionally, the two cited drugs were also determined in the presence of dipyrone active metabolite 4-aminoantipyrine using diclofenac as an internal standard in bovine urine. The linearity range was found to be (15–75 µg/mL) for dipyrone, (2.5–60 µg/mL) for hyoscine, and (2.5–60 µg/mL) for 4-aminoantipyrine with linearity regression R (0.9999–0.9998). The LLOQ (15, 2.5, 2.5 µg/mL), LQC (45, 7.5, 7.5 µg/mL), MQC (55, 25, 25 µg/mL), and HQC (60, 50 50 µg/mL) were determined for dipyrone, hyoscine and 4-aminoantipyrine, respectively. UV detection was carried out at 220 nm. The method was validated according to the ICH guidelines, as well as according to FDA guidelines for determining both drugs in bioanalytical matrices and both proved accuracy and precision. A statistical comparison was made between the results obtained and those obtained by the reported method, showing no significant difference in accuracy and precision at p = 0.05. The suggested method was proved eco-friendly through a greenness assessment using two different tools (The analytical eco-scale scored 83, and the AGREE-Analytical Greenness Metric approach scored 0.83). The suggested method can be used in the routine work of quality control labs, screening for drug abuse, and ensuring clean sport for horse racing.


List of tables
Table S1: System suitability parameters of the developed HPLC method for the determination of DIP, HBB and their impurities.
Table S2: Robustness of the proposed HPLC method for determination of DIP and HBB and some impurities.
Table S3: System suitability parameters of the developed HPLC method for the determination of DIP, HBB and AMP in urine Table S4: Stability results of simultaneous determination of DIP, HBB and AMP in bovine urine by the proposed method Table S5: Statistical comparison of the results obtained by the proposed method and the reported method for the determination of DIP and HBB in urine Table S6: Penalty points used in the Analytical eco-scale tool for the assessment of the greenness of the method The method validation was performed according to the ICH guidelines [1] for the analysis of the two drugs DIP and HBB in their pure form and their combined dosage form by preparing mixtures of the two drugs containing 1.00, 5.00, 15.00, 25.00, 35.00 and 40.00 µg/mL for DIP and 2.50, 10.00, 20.00, 30.00, 40.00 and 50.00 µg/mL for HBB each concentration was injected three times and chromatographed using the optimum condition, the calibration curve was plotted for each drug using the peak area versus the corresponding concentration.The limit of detection (LOD) and limit of quantification (LOQ) were calculated by equations (S1, S2):

List of Figures
Where S/N is the signal-to-noise ratio The method specificity is the ability to separate the three drugs with peaks of good resolution without any interference from the pharmaceutical preparation's excipients.
The inter-day and intra-day precisions were evaluated using a mixture of the two drugs using five concentrations (2,10,20,30,35 ug/mL) of DIP and (5,15,25,45,50 ug/mL) of HBB.Regarding the intra-day precision, each concentration was injected in triplicate on the same day, while in the inter-day precision, each concentration was injected on three consecutive days.The results were evaluated using percentage relative standard deviation (% RSD).
The accuracy of the method was determined by using a mixture of the two drugs of concentrations Method robustness is identified as the proposed method's ability to be unaffected by deliberate changes in the chromatographic conditions.The studied conditions were the pH value of the mobile phase, flow rate, and the percentage of aqueous: organic solvent in the mobile phase.The results were evaluated by the %RSD.
The method's system suitability parameters, including retention factor, resolution, selectivity factor, capacity factor, theoretical plate count, and the height equivalent to each theoretical plate, were tested.

S.1.2. Bioanalytical method validation
Bioanalytical validation of the suggested method was done according to the FDA guidelines [2] to ensure that the suggested method is satisfactory for its intended purpose, starting with the sample preparation step and continuing through the process.

S.1.2.1. Linearity and range
The method's linearity was investigated using nine non-zero samples of DIP, HBB, and AMP spiked in urine.In the range of (15-75 µg/mL) for DIP, (2.5-60 µg/mL) for HBB and (2.5-60 µg/mL) for AMP.In addition, six blank urine samples were analyzed to ensure the absence of any interferences.
The calibration curves were plotted using each analyte concentration against the corresponding relative peak area after dividing each drug's peak area by the peak area of the IS DIC to get relative peak areas.The acceptance criterion of the calibration curve is the regression coefficient (R²) with a value of 0.99 or better, and each calculated standard concentration must be within 15.0 % deviation from the nominal value except for LLOQ, where the acceptable 100 deviation was 20.0 %.

S.1.2.2. Lower limit of quantification (LLOQ)
It is the lowest concentration in the calibration curve, with accuracy from 80.0 to 120.0 %.At the same time, its % RSD is less than 20.0 %.

S.1.2.3. Selectivity
It was evaluated by injecting six blank urine samples obtained from different cows to ensure that the analytes were not interfered with by the urine matrix.

S.1.2.4. Accuracy and precision
Accuracy was evaluated by % R of the QC samples, and the accepted range was as mentioned in section S.1.2.1.For intra-day precision, QC samples were analyzed six times on the same day, and each QC sample was analyzed six times on six different days for inter-day precision.The results were evaluated by % RSD, and the accepted range was as mentioned in section S.1.2.1.

S.1.2.5. Extraction recovery
The extraction recoveries of DIP, HBB, and AMP from urine were examined by comparing the mean peak area of triplicates of four unextracted samples of drugs (LLOQ, LQC, MQC and HQC) to peak areas of the same concentrations with prepared spiked urine (14.00, 45.00, 55.00 and 60.00 µg/mL) for DIP and (2.50, 7.50, 25.00 and 50.00 µg/mL) for HBB and AMP and % R was calculated as in equation (S3).

S.1.2.6. Stability
QC samples were injected in triplicates for all conditions and then compared to freshly prepared samples.
Short term: LLOQ, LQC, MQC, and HQC samples were thawed at room temperature and kept at this temperature for 4-6 hours, then analyzed.
Post-preparative: The stability of the prepared samples was investigated by keeping three replicates of the LLOQ, LQC, MQC, and HQC samples at room temperature for 24 hours.
Long-term stability was examined by storing three aliquots of LLOQ, LQC, MQC, and HQC concentrations of the studied drugs at -20 °C for 30 days, then thawing them at room temperature and analyzing them.
Freeze and thaw: Three aliquots at each of LLOQ, LQC, MQC, and HQC samples were stored at -20°C for 24 hours and thawed at room temperature.When completely thawed, the samples were refrozen for 24 hours under the same conditions.The freeze and thaw cycles were repeated three times and then analyzed.The results were evaluated by % deviation from the freshly prepared samples using equation (S4), where the deviation should be ± 15 %.
Also, the method was selective due to its ability to separate the two drugs in the presence of their impurities and preservatives without any interference.
The results in Table S2 showed that the method was robust due to its ability to resist small changes in the chromatographic conditions.Finally, the method passes the system suitability parameters in Table S1.

S.2.3.3. Selectivity
The method was selective, as the blank urine samples were chromatographed without interferences.In addition, it is possible to separate the two main drugs along with DIP metabolite AMP and DIC as IS with sharp peaks without any interferents from the urine proteins, as shown in (Fig. 4).

S.2.3.4. Accuracy and precision
The method was accurate and precise, as shown in Table 3.

S.2.3.5. Extraction recovery
The extraction protocol of the three drugs from the urine samples showed high efficiency, as represented in Table 4.

( 2 ,
10,20,30,35 ug/mL) of DIP and (5,15,25,45,50 ug/mL) of HBB and the results were calculated as %R.Accuracy (%) = Actual Conc.(practical Conc.)/TheoreticalConc.× 100 LLOQ, LQC, MQC, and HQC samples were thawed at room temperature and kept at this temperature for 4-6 hours, then analyzed b The stability of the prepared samples was investigated by keeping three replicates of the LLOQ, LQC, MQC, and HQC samples at room temperature for 24 hours.c Long-term stability was examined by storing three aliquots of LLOQ, LQC, MQC, and HQC concentrations of the studied drugs at -20 °C for 30 days.Then, they were thawed at room temperature and analyzed.d three aliquots at each of LLOQ, LQC, MQC, and HQC samples were stored at -20°C for 24 hours and thawed at room temperature.When completely thawed, the samples were refrozen for 24 hours under the same conditions.The freeze and thaw cycles were repeated three times and then analyzed.The concentrations used for measuring the stability of DIP (15,45,55 and 60 ug/mL), HBB, and AMP (2.5, 7.5, 25and 50 µg/mL)

Table S5 : Statistical comparison of the results obtained by the proposed method and the reported method [4] for the determination of DIP and HBB in urine
Values between parenthesis are the theoretical value of t and F at P= 0.05 and n= 5 ** HPLC method using C18 (250 mm x 4.6 mm) mobile phase composed of (water: methanol, 50:50) pH adjusted to 7 using triethylamine and trifluoroacetic acid Uv detection 210 nm. *